The Y498T499-SARS-CoV-2 Spike (S) protein variant interacts with rat ACE2 but does not infect or induce responses in the rat lung when delivered as a S-protein pseudotyped lentivirus

The rat is a useful laboratory model for respiratory disease, and SARS-CoV-2 proteins such as the spike (S) protein can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change described in the S-protein of the mouse adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lung. Using a real-time S-ACE2 quantitative fusion assay, ancestral S-protein fuses with human, but not rat ACE2. The QP-YT S-protein retains ability to fuse with human ACE2, and interacts with rat ACE2 in the fusion assay and using a S-protein pseudotyped lentivirus infection system. L452R S-protein did not bind to rat ACE2. Although rat lower lung contains both ACE2 and TMPRSS2 target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration, or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed. Analysis of the amino acid changes across the S-ACE2 interface highlights the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2 and the QP-YT S-protein variant can bind to rat ACE2 but this does not result in infection or stimulate responses in the lung. Further amino acid changes in S-protein could enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving inflammation in the lung.